Group B Streptococci (GBS) are the leading cause of neonatal sepsis and meningitis. Maternal IgG antibodies to the GBS polysaccharide (PS) protect the neonate from invasive GBS disease. Such antibodies are deficient in mothers of infants who develop GBS disease. A case control study was designed by NICHD to identify amounts of antibody needed for protection against type IIII. Paired maternal and cord sera from different regions of the United States were analyzed in our laboratory for presence of naturally occurring GBS type III PS antibodies. Measurements of the antibody concentrations for GBS type III have proven to be more challenging than our earlier work with type Ia. Earlier work in our laboratory showed that conjugation of the GBS III PS to albumin changes some of the PS epitopes. We therefore used the native GBS type III polysaccharide that we purified by ion exchange chromatography to be free of the contaminating group B antigen. We have now completed our blinded analysis of over 140 sera in the case control study, and the statistical evaluation has been completed by our colleagues at NICHD. Conjugate vaccines utilize a carrier protein as a vehicle to convert the T-independent PS to a T-dependent protein-PS conjugate, and thus afford protection to an otherwise PS-non-responding population such as infants and the elderly. In our research, we have investigated issues related to the chemistries of conjugate and their impact upon vaccine immunogenicity. Current methods for synthesis and manufacturing of polysaccharide-protein conjugate vaccines employ conjugation reactions with low efficiency (about 20%). This means that up to 80% of the added activated polysaccharide (PS) is lost. In addition, inclusion of a chromatographic process for purification of the conjugates from unconjugated PS is required. We have developed a new conjugation method that utilizes hydrizide groups on one reactant to react with aldehyde or cyanate groups on the other reactant with an improved yield (50 to 65%), and thus simplify the purification process of conjugate product by avoiding the chromatographic procedure. Meningococcal group C and group A polysaccharides conjugated to tetanus toxoid were up to 100 times more immunogenic in mice than the native polysaccharides. The physical and chemical properties of these conjugates have been investigated using procedures developed or modified in the LBP. Most laboratories that produce conjugate vaccines examine conjugation efficiency by estimation of the remaining unbound PS, but due to the high efficiency conjugation process utilized in the LBP. We developed a method to precipitate deoxycholate protein complexes largely free of unconjugated PS, and thereby determine protein bound PS. Results of this analysis were confirmed by use of Sephacryl S300 chromatography and immunodetection individually of both the tetanus toxoid carrier and the meningococcal polysaccharide. Immunological studies of pneumococcal polysaccharide antibodies. Pneumococcal polysaccharide antigens induce type-specific antibodies that enhance opsonization, phagocytosis, and killing of pneumococci by leukocytes and other phagocytic cells. The licensed pneumococcal conjugate vaccine, Prevnar, contains polysaccharides from the seven most prevalent serotypes that cause invasive pneumococcal disease in children. We have investigated antibodies present in commercial IGIV preparations to the serotypes present in Prevnar. We have also examined the sub-classes of IgG (IgG1, IgG2, and IgG3) in IVIG (Immunoglobulin) preparations from 3 different manufacturers (in collaboration with B. Golding laboratory) were assayed for antibody levels in 5 Pn serotype (4,6B, 9V, 14 and 19F) by ELISA and functional activity of these antibodies were examined by opsonophagocytosis assay. There was a predominance of IgG2 to the different serotypes tested and these antibodies were functional by opsonophagocytosis assay. We have investigated new methods for preparation of conjugates applicable to the pneumococcal polysaccharides. Conjugate vaccines utilize a carrier protein as a vehicle to convert the T-independent PS to a T-dependent protein-PS conjugate, and thus afford protection to an otherwise PS-non-responding population such as infants and the elderly. In our research, we have investigated issues related to the chemistries of conjugate and their impact upon vaccine immunogenicity. Current methods for synthesis and manufacturing of polysaccharide-protein conjugate vaccines employ conjugation reactions with low efficiency (about 20%). This means that up to 80% of the added activated polysaccharide (PS) is lost. In addition, inclusion of a chromatographic process for purification of the conjugates from unconjugated PS is required. We have developed a new conjugation method that utilizes hydrizide groups on one reactant to react with aldehyde or cyanate groups on the other reactant with an improved yield (50 to 65%), and thus simplify the purification process of conjugate product by avoiding the chromatographic procedure. Meningococcal group C and group A polysaccharides conjugated to tetanus toxoid were up to 100 times more immunogenic in mice than the native polysaccharides. The physical and chemical properties of these conjugates have been investigated using procedures developed or modified in the LBP. Most laboratories that produce conjugate vaccines examine conjugation efficiency by estimation of the remaining unbound PS, but due to the high efficiency conjugation process utilized in the LBP. We developed a method to precipitate deoxycholate protein complexes largely free of unconjugated PS, and thereby determine protein bound PS. Results of this analysis were confirmed by use of Sephacryl S300 chromatography and immunodetection individually of both the tetanus toxoid carrier and the meningococcal polysaccharide. Immunity to group B streptococcal disease. Group B Streptococci (GBS) are the leading cause of neonatal sepsis and meningitis. Maternal IgG antibodies to the GBS polysaccharide (PS) protect the neonate from invasive GBS disease. Such antibodies are deficient in mothers of infants who develop GBS disease. A case control study was designed by NICHD to identify amounts of antibody needed for protection against type IIII. Paired maternal and cord sera from different regions of the United States were analyzed in our laboratory for presence of naturally occurring GBS type III PS antibodies. Measurements of the antibody concentrations for GBS type III have proven to be more challenging than our earlier work with type Ia. Earlier work in our laboratory showed that conjugation of the GBS III PS to albumin changes some of the PS epitopes. We therefore used the native GBS type III polysaccharide that we purified by ion exchange chromatography to be free of the contaminating group B antigen. We have now completed our blinded analysis of over 140 sera in the case control study, and the statistical evaluation has been completed by our colleagues at NICHD. Immune responses of pneumococcal conjugate vaccines using common pneumococcal protein carriers. Protection against pneumococcal infection depends on the presence of antibodies against capsular polysaccharides (PSs). These serotype-specific antibodies facilitate phagocytosis to destroy invading bacteria. The spleen is an important organ in the immune response to pneumococci, since it contains both antibody-producing B-cells and phagocytes. The 23-valent pneumococcal PS vaccine is effective against bacteremic pneumococcal disease in adults and is cost-effective for routine immunization of the elderly. However, the PS vaccine is not sufficiently immunogenic in infants and young children. Chemical coupling of a PS to a carrier protein to form a glycoconjugate greatly improves the immunogenicity of a PS. The 7-valent pneumococcal conjugate vaccine, Prevnar, appears to be highly effective in preventing invasive disease in young children and to have a significant impact on otitis media. Studies were conducted in our laboratory to evaluate the avidity, antibody response and opsonophagocytic activity of serum antibodies to the pneumococcal type 9V PS conjugated to inactivated pneumolysin (Ply) or autolysin (Aly) and in the 7-valent PS-CRM197 conjugate vaccine. The effect of priming mice with the 7-valent conjugate followed by a booster dose of 23-valent PS vaccine was also examined. Compared to 9V PS alone or non-immunized controls, the serum concentrations of 9V PS IgG and IgM antibodies were significantly higher in mice immunized with 9V PS-Ply, 9V PS-Aly conjugates, or 7-valent conjugate vaccine at 0, 2, and 4 weeks, 7-valent conjugate followed by 23-valent PS vaccine compared to 9V PS alone. Thus, priming mice with polyvalent conjugate vaccine induced high 9V PS antibody response in contrast to the group primed with polyvalent PS vaccine. The duration of antibody response in mice was studied in mice immunized with various conjugate vaccines and boosted with 23-valent PS vaccine. The antibody response was highest at 4 weeks after the injection, increasing 7-10 fold compared to the group that received 9V PS alone. The high IgG Ab response was maintained for over 12 weeks after final injection. Similar results were also observed in 9V PS IgM Ab response. In addition, high IgG and IgM Abs were maintained for over 8 weeks after final injection in mice given a booster dose of 23-valent PS vaccine. The avidity indeces of IgG antibody were highest in mice immunized twice with the 7-valent conjugate vaccine, and combined immunization with 7-valent conjugate then 23-valent PS vaccine, followed by monovalent 9V PS-protein conjugates. Mice immunized with 9V PS alone exhibited the lowest avidity. Similar results were observed in the avidity of IgM antibody. The 9V PS-Ply and 9V PS-Aly immunized group showed the opsonophagocytic titers of 16 compared to 4 in 9V PS-immunized and complement control groups. Mice immunized twice with 7-valent conjugate or conjugate vaccine followed by 23-valent PS vaccine showed titers of 32 and >64 respectively. These results indicate that monovalent and 7-valent conjugate vaccines can stimulate effective functional activity that correlated with protective immunity against pneumococcal infection. This project incorporates FY2002 projects 1Z01BJ002014-09, 1Z01BJ002021-07, and 1Z01BJ002029-05.